Interlab

Synthetic biology and all the engineering disciplines need to be reliable and repeatable, this year iGEM TecCEM 2018 is one of the teams that participated in the Fifth International InterLaboratory Measurement Study. In this page, we report the results obtained during all protocols.

Competent cell - transformation DH5-alpha

Transformations of all devices were performed, and the resulting transformant strains were plated on LB agar, with 35 μg/mL chloramphenicol. Two colonies per plate were separated into two independent cultures.

Verification

We verified the presence and size of the device plasmids in the transformant strains by running an agarose gel electrophoresis.

Table 1. Devices from the Interlab Study with their pait number and respective size.

Device	Part Number	Size (bp)
Negative Control	BBa_R0040	2124
Positive Control	BBa_I20270	2989
Device 1	BBa_J364000	2988
Device 2	BBa_J364001	2988
Device 3	BBa_J364002	2988
Device 4	BBa_J364007	2988
Device 5	BBa_J364008	2988
Device 6	BBa_J364009	2988

Calibration Protocol

The main objective of this calibration LUDOX protocol is to use LUDOX CL-X to get the conversion factor between absorbance (Abs ₆₀₀) from the plate reader and the OD₆₀₀ measurement.

Table 2. OD₆₀₀ Reference Point - LUDOX Protocol.

	LUDOX CL-X	h1O
Replicate 1	0.059	0.033
Replicate 2	0.061	0.033
Replicate 3	0.062	0.033
Replicate 4	0.060	0.033
Arith. Mean	0.061	0.033
Corrected Abs₆₀₀	0.028
Reference OD₆₀₀	0.063
OD₆₀₀/Abs₆₀₀	2.291

Particle standard curve - Microsphere Protocol

The aim of this protocol is to establish a standard curve from a microsphere solution. These microspheres have similar size and optic properties to bacterial cells, therefore, they may be used to calibrate.

After preparing the Microsphere Stock Solution, serial dilutions were made in a 96-well plate as instructed. To achieve reliable results, the plate reader was set to shake the samples just before reading them. The reads were registered in the given Excel file and are shown below.

Figure 1. Standard curve for particle count/100 uL

Fluorescence standard curve - Fluorescein Protocol

Following the protocol for the fluorescence standard curve using fluorescein, we prepared the corresponding reactants and the requested serial solutions in the 96-well plate.

Interlab plot 3 — Figure 2. Standard curve for fluorescein concentration

Cell measurement Protocol

Table 3. Absorbance and fluorescence at hour 0 and hour 6.

	Hour 0		Hour 6
	Abs₆₀₀ Raw Readings	Fluorescence Raw Readings	Abs₆₀₀ Raw Readings	Fluorescence Raw Readings
Negative Control	0.078375	153.5	0.46225	170.125
Positive Control	0.074	220.125	0.428125	1926.75
Device 1	0.0715	439.125	0.241	4319.5
Device 2	0.079125	266.625	0.442375	2148.5
Device 3	0.075625	151.125	0.430375	207.625
Device 4	0.074375	601.5	0.347625	5545.875
Device 5	0.075625	335.875	0.16425	2039
Device 6	0.077	227.125	0.45325	1151
LB + Chlor (Blank)	0.053375	158.625	0.051125	177.5

After performing all three calibration measurements, we proceeded to execute the cell measurement protocol, previously having the overnight cell culture of each device and control. We use the same plate and volume that we used in the calibration protocol, also the same settings in order to make valid the measurement.

We defined the difference between the measurement of the absorbance and fluorescence, starting in the hour 0 until the hour 6.

Colony Forming Units per 0.1 OD₆₀₀ E. coli cultures

To preparing the starting samples we diluted overnight cultures of both positive and negative controls to the linear detection range of the plate reader. A duplicate of each culture was placed on the 96-well plate, as well as a blank. After measuring absorbance, cultures were diluted further to reach an OD₆₀₀=0.1. A triplicate of each dilution was placed as instructed. The absorbance measurements were confirmed and used to prepare the respective dilutions for 12 starting samples at 0.1, six for each control. We diluted the cultures to a final OD₆₀₀ of 0.1 and performed serial dilutions by transferring 100 μL to the next tube. The dilutions 3, 4, and 5 were used to spread 100 μL in LB+Cam plates. After an overnight incubation, we counted the colonies in each of the plates, being many of them uncountable. The counted colonies are shown below.

Table 4. Colony count.

Plate	Colony count
Plate	Dilution 3	Dilution 4	Dilution 5
A-1	564	417	49
A-2	1120	286	64
A-3	1624	360	78
B-1	984	349	53
B-2	1224	277	55
B-3	1156	369	45
A+1	1340	90	5
A+2	812	243	16
A+3	1052	71	14
B+1	1146	180	25
B+2	1116	250	21
B+3	888	138	36

Conclusion

Devices 2 and positive control exhibited a slow growth rate, hence, we had issues when waiting for cultures to reach a desired absorbance, as they became out of phase. Several teams we contacted had experienced similar situations. Despite that, InterLab is useful tool to evaluate the reliability of the measurements performed in the laboratory. We think it represents a means to demonstrate the technical quality of team members.

Team:TecCEM/InterLab